The two-dose BNT162b2 (Pfizer-BioNTech) vaccine has demonstrated high efficacy against COVID-19 disease in clinical trials of children and young people (CYP). Consequently, we investigated the uptake, safety, effectiveness and waning of the protective effect of the BNT162b2 against symptomatic COVID-19 in CYP aged 12–17 years in Scotland.The analysis of the vaccine uptake was based on information from the Turas Vaccination Management Tool, inclusive of Mar 1, 2022. Vaccine safety was evaluated using national data on hospital admissions and General Practice (GP) consultations, through a self-controlled case series (SCCS) design, investigating 17 health outcomes of interest. Vaccine effectiveness (VE) against symptomatic COVID-19 disease for Delta and Omicron variants was estimated using a test-negative design (TND) and S-gene status in a prospective cohort study using the Scotland-wide Early Pandemic Evaluation and Enhanced Surveillance of COVID-19 (EAVE II) surveillance platform. The waning of the VE following each dose of BNT162b2 was assessed using a matching process followed by conditional logistic regression.Between Aug 6, 2021 and Mar 1, 2022, 75.9% of the 112,609 CYP aged 16–17 years received the first and 49.0% the second COVID-19 vaccine dose. Among 237,681 CYP aged 12–15 years, the uptake was 64.5% and 37.2%, respectively. For 12–17-year-olds, BNT162b2 showed an excellent safety record, with no increase in hospital stays following vaccination for any of the 17 investigated health outcomes. In the 16–17-year-old group, VE against symptomatic COVID-19 during the Delta period was 64.2%  at 2–5 weeks after the first dose and 95.6% (77.0–99.1) at 2–5 weeks after the second dose. The respective VEs against symptomatic COVID-19 in the Omicron period were 22.8% (95% CI -6.4–44.0) and 65.5% (95% CI 56.0–73.0). In children aged 12–15 years, VE against symptomatic COVID-19 during the Delta period was 65.4% (95% CI 61.5–68.8) at 2–5 weeks after the first dose, with no observed cases at 2–5 weeks after the second dose. The corresponding VE against symptomatic COVID-19 during the Omicron period were 30.2% (95% CI 18.4–40.3) and 81.2% (95% CI 77.7–84.2). The waning of the protective effect against the symptomatic disease began after five weeks post-first and post-second dose.During the study period, uptake of BNT162b2 in Scotland has covered more than two-thirds of CYP aged 12–17 years with the first dose and about 40% with the second dose. We found no increased likelihood of admission to hospital with a range of health outcomes in the period after vaccination. Vaccination with both doses was associated with a substantial reduction in the risk of COVID-19 symptomatic disease during both the Delta and Omicron periods, but this protection began to wane after five weeks.UK Research and Innovation ; Research and Innovation Industrial Strategy Challenge Fund; Chief Scientist's Office of the Scottish Government; Health Data Research UK; National Core Studies – Data and Connectivity. We searched PubMed on March 31, 2022, using the search terms “(COVID-19 OR SARS-CoV-2) AND vaccin* AND (children OR adolescent* OR young people)” with no restriction on language. Among 4,723 studies, we found 66 titles relevant to our areas of interest, namely vaccine uptake, safety, effectiveness, or waning. From those studies, it was clear that the two-dose BNT162b2 vaccine safety, immunogenicity and efficacy against COVID-19 in CYP has been initially demonstrated by the phase 3 clinical trials sponsored by the manufacturers – firstly those aged 16–17, then 12–15, and most recently 5–11 years. In China, Israel, the United States and England, the uptake of the first dose among 12–17 year-olds was typically above 50%, while it was slightly lower for the second dose and in younger age groups. The analyses of safety reported myocarditis as a rare outcome in high-school boys following the second dose of the BNT162b2 vaccine. Sleep irregularities were also reported as a possible adverse effect. The effectiveness of the first dose against symptomatic COVID-19 disease for the Delta variant in 12–17-year-olds typically ranged between 55–65% and of the second dose between 87–99%. The effectiveness against more severe outcomes requiring hospitalisation was better than against symptomatic infections. A few reports on VE against the Omicron variant suggested that VE is considerably lower in comparison to the Delta variant. Analysis of the waning of the protective effect showed a relatively rapid decline in effectiveness against symptomatic disease, within only a few weeks. VE against severe forms of COVID-19 may last longer, but this still needs to be confirmed.To our knowledge, this is the first study of COVID-19 vaccine uptake, safety, VE and waning against symptomatic disease after two doses for an entire nation, addressing both the Delta and the Omicron variant period. Among 16–17 year-olds in Scotland, 75.9% received the first dose and 49.0% the second dose; among 12–15 year-olds, the uptake was 64.5% and 37.2%, respectively. The safety record was encouraging, with none of the 17 investigated health outcomes showing an increased rate of hospitalisation following vaccination. In the 16–17-year-old group, VE against symptomatic COVID-19 during the Delta period was 64.2%  at 2–5 weeks after the first dose and 95.6% (77.0–99.1) at 2–5 weeks after the second dose. The respective VEs against symptomatic COVID-19 in the Omicron period were 22.8% (95% CI -6.4–44.0) and 65.5% (95% CI 56.0–73.0). In children aged 12–15 years, VE against symptomatic COVID-19 during the Delta period was 65.4% (95% CI 61.5–68.8) at 2–5 weeks after the first dose, with no observed cases at 2–5 weeks after the second dose. The corresponding VE against symptomatic COVID-19 during the Omicron period were 30.2% (95% CI 18.4–40.3) and 81.2% (95% CI 77.7–84.2). The waning of the protective effect started after five weeks post-first and post-second doses.High levels of BNT162b2 coverage can be achieved in CYP, with subsequent effectiveness against symptomatic COVID-19. However, protection against symptomatic disease is short-lived. We found no increase in hospitalisation following vaccination for any of the adverse health outcomes of interest.Alt-text: Unlabelled box,,,The early research to characterise SARS-CoV-2 infection and COVID-19 disease in children and young people (CYP) was initially focused on the clinical presentation, patterns of spread, viral load, diagnosis and treatment, and the arguments related to vaccination of CYP which we recently reviewedWe developed a protocol for evaluating the “real-world” effectiveness of the COVID-19 vaccination programme.Information on the cohorts used for each analysis, primary data sources, data diagram and key dates relevant to the time period of different analyses are presented in Supplementary Table S2 and Supplementary Figures S1–S6.48The analysis of the vaccine uptake was based on information gathered from the Turas Vaccination Management Tool, inclusive of the date Mar 1, 2022 . The numUptake figures include 6,865 CYP vaccinated before the period of the study . This is a sub-sample of CYP with specific individual or household vulnerabilities, who were offered vaccination before these dates and also followed a different schedule of recommended follow-up doses (detailed in Supplementary Table S2). Vulnerabilities included conditions such as a history of chronic respiratory disease, chronic heart conditions, chronic conditions of the kidney, liver or digestive system, chronic neurological disease, endocrine diseases, dysfunction of the spleen, serious genetic abnormalities, pregnancy, and different forms of immunosuppression (detailed in Supplementary Tables S2 and S3).The safety of the BNT162b2 vaccine in CYP was evaluated using national data on hospital admissions and General Practice (GP) consultations, through a self-controlled case series (SCCS) design. This analysis is not restricted to EAVE II data, and it includes all vaccinated 12–17-year-olds in Scotland, including those who were at higher risk of developing severe COVID-19. More than 95% of children aged 12–17 years are included in the EAVE II data, and the missing ones are likely to be new arrivals in Scotland. Twenty-nine potential adverse events of special interest (AESI) were chosen for inclusion in this study based on the list of outcomes recommended for monitoring by the World Health Organization,The number of hospital stays that occurred in a baseline period  were calculated for each individual, for each vaccine dose number and health condition . A SCCS To study VE and examine waning, we used the Early Pandemic Evaluation and Enhanced Surveillance of COVID-19 database comprising linked vaccination, primary care, and reverse transcriptase-polymerase chain reaction (RT-PCR) testing for children and young people (aged 16–17 and 12–15 years) in Scotland.The estimates are based upon a national prospective cohort study with test negative cohort design, where vaccination records are linked to Electronic Communication of Surveillance in Scotland (ECOSS) for an endpoint of COVID-19 RT-PCR positive test and a notion of the presence of symptoms. Symptom status is based upon self-reported information provided at the time of completion of the online test request form. Details on ECOSS data are available in Supplementary Table S2.In this study, two periods were defined based on the dominant SARS-CoV-2 variant circulating in communities at the time, Delta and Omicron.This analysis includes the BNT162b2 vaccine for two doses. In Scotland, all children were on a 2-dose schedule, apart from immunocompromised, who are on a 3-dose schedule. A matched test-negative design (TND) was used to estimate the odds ratios comparing unvaccinated at the time of test with post-first and post-second dose vaccination periods measured in weeks. Positive tests were matched with up to 5 controls using the date of test, age and location, with a conditional logistic regression model stratified by the positive case and individually matched controls. Positive tests which could not be matched on these variables were discarded. The conditional logistic regression model included vaccine status, sex, socioeconomic status measured by quintiles of the Scottish Index of Multiple Deprivation (SIMD), whether the CYP had previously tested positive at any time before the specimen date, the number of tests previously taken, and the following QCovid risk conditions: Blood Cancer, Cerebral Palsy, Epilepsy, Fracture, Severe Mental Illness, Learning Disability, Congenital Heart Defect, and the number of other QCovid risk conditions the individual has. VEs were measured relative to the unvaccinated. Vaccine effectiveness is defined as “1 – odds ratio” from the conditional logisitic regression. Details on statistical modelling are presented in Supplementary Table S2. They include the full set of variables used to analyse the waning of vaccine effectiveness.We provided STROBE and RECORD statements in Supplementary Table S7. The availability of data and code is explained in the acknowledgements.The EAVE II Public Advisory Group reviewed the analysis design for this project. Among their suggestions relevant to this work, we complied by explaining any excluded groups or aggregations should and separately reporting vaccine uptake in 16–17 year-olds.The funders did not have any influence on the study design, analyses, interpretation or decision to publish.There were 350,300 CYP aged between 12–17 years who were resident in Scotland during the period of study between Aug 6, 2021 and Mar 1, 2022 . The 16–p=0.070) (Supplementary Table S5). We examined the associations of exposures with hospital admissions for poisoning in CYP as a negative control outcome. We found no increased risk of poisoning in the 1–90 days following vaccine exposure (Supplementary Tables S4 and S5).Among 12–17-year-olds, 13 of the 17 studied health outcomes had fewer than five hospital stays in their corresponding risk periods following both first and second dose BNT162b2 vaccination . For thoFor the Delta period, among 16–17-year-olds the first dose led to a 64.2% (95% CI 59.7-68.5) reduction in symptomatic COVID-19 in the period 2–5 weeks after vaccination, but the effect waned to 34.8% (95% CI 20.9-46.2) after 10-13 weeks a. HoweveIn the 16–17 year olds, VE during the Omicron period was smaller than during the Delta period b. The fiAmong 12-15 year-olds for the Delta period, the first dose led to a 65.4% (95% CI 61.5-68.8) reduction in the period 2-5 weeks after vaccination, but the effect waned and dropped to 44.5% (95% CI 34.1-53.3) after 10-13 weeks c. HoweveWhen examining VE during the Omicron period in 12-15-year-olds, the first dose led to a 30.2% (95% CI 18.4-40.3) reduction 2-5 weeks after vaccination, but this effect waned to 16.9% (95% CI 8.7-24.4) after 10-13 weeks d. The seThis study showed that high levels of BNT162b2 vaccine coverage can be achieved among CYP through national vaccination programmes. Once licensed for 16–17-year-olds, the first dose of BNT162b2 reached coverage of above 50% within a month. However, the uptake was much slower thereafter, reaching 75.9% by Mar 1, 2022. A similar pattern was observed among the 12–15-year-olds, where the uptake was also rapid within the first month of the programme, but it eventually reached 64.5% and stalled. By the end date of this study, the uptake was lower for the second dose and among the younger age group at that point in time.,,57There are many possible contributing factors to this finding: some children may have not yet been eligible for their second dose before the end date of this study, due to the required interval between doses. This is particularly likely to apply to the younger age group who received their offer of the first dose at a later date, or the second dose may have been delayed due to symptomatic COVID-19 following the first dose. Other possible reasons include the initial offer being a single dose schedule potentially influencing the perception of the need for a second dose; then, the perceived rarity of severe illness in children; furthermore, there could have been increased awareness of reports of sub-optimal vaccine prevention of the spread of infection, of possible side-effects, and of the waning of the protective effect.Vaccination is overall safe and effective in protecting against symptomatic COVID-19 disease the first few months post-vaccination. However, the protection against symptoms was short-lived during the Delta and Omicron periods in Scotland. In both 16–17 and 12-15 year-olds, the waning of the effectiveness during the Delta period was detectable from week 6 following the first dose. However, the effectiveness remained higher for a longer time period following the second dose. In both age groups, the effectiveness of the first dose during the Omicron period was modest and it waned rapidly after week 5. Furthermore, the second dose offered stronger and more durable protection, but it also typically waned from week 6 following the second dose. For adults, protection against severe outcomes was higher and longer-lasting than protection against symptomatic infection.,,,,The UK medicines regulator, the Medicines and Healthcare products Regulatory Agency (MHRA), has assessed the BNT162b2 vaccine and declared it safe for 12–17 year-olds. This followed a rigorous review of the safety, quality and effectiveness of these vaccines among CYP of that age.,In Scotland, there were very few hospitalisations for the 17 health outcomes in 12–17 year-olds following the first and second dose of the BNT162b2 , with noOur study has several strengths. It is a national-level study. This ensured sufficiently large numbers to observe effects and patterns. Previous studies using the EAVE II platform have demonstrated the usefulness of integrated data from multiple sources and enabled precise analyses of the pandemic almost in real-time,There are some caveats and limitations specific to COVID-19 research in CYP. First, there was a sub-group of CYP in Scotland  who were in the category of particularly vulnerable and at-risk from COVID-19. They were vaccinated in parallel with adults in Scotland, and before most CYP, using vaccines that were licensed for adults at the time. Therefore, they were not comparable to other CYP because of different inherent risks of COVID-19 complications, different time periods, exposure to different SARS-CoV-2 variants, and likely smaller average number of possible exposures because they were shielded. They also received different vaccines and dosages. This is why we did not include this relatively small sub-group of CYP in the VE analyses, but we retained them for safety analyses.Moreover, a recent previous SARS-CoV-2 infection may have delayed vaccination and impacted vaccine uptake.In the study of safety, a preceding hospitalization during the period 75-to-15-days before the first dose of vaccine may have influenced the timing to receive vaccination, and thereby bias the SCCS data, although this is mainly a theoretical concern as such events were rare. The use of negative controls in SCCS, both at exposure or outcome side, has been proven to be a valuable approach to evaluate potential biases and many assumptions required to run a reliable SCCS.Another limitation is that the assumptions made to determine dominant variant time periods may incorrectly assign COVID-19 cases to a given variant, thus impacting vaccine effectiveness calculations. Also, it was not possible to distinguish between specific sub-variants within the Omicron variant because only a subset of samples was sent for Whole Genome Sequencing (WGS). Furthermore, the second dose of the vaccine in both 16–17-year-olds and 12-15-year-olds was given with a delay of several weeks after the first dose see . In paraThe outcome of TND was defined by the positive RT-PCR test and a provision in the dataset that the disease was symptomatic in a given test-positive case. A potential caveat in choosing the study design was that children were also tested twice a week at their schools using lateral flow tests (LFT). We decided to exclude all LFT results from the analyses because children are more likely to report positive than negative tests which could have introduced bias.The negative VE estimates in the Omicron period could have several plausible explanations. First, this could be due to behavioural confounding, where vaccinated CYP begin to act more freely and take less caution. This is how they can eventually get more symptomatic infections, as a net result of modest vaccine protection against symptomatic infection, waning of the effectiveness, and their behaviour. Initially, the vaccine effect is still dominant, but as it wanes over time, the behaviour effect might cause negative VE. Second, at this stage of the pandemic it is plausible that there were more CYP who acquired natural immunity among the unvaccinated group than among the vaccinated. Natural immunity may be either more robust, more durable, or both, in comparison to vaccine induced immunity against the omicron variant, leading to negative VE over time.,We also planned to study VE against severe COVID-19 outcomes.,In this paper, we addressed several important research questions on CYP in a single publication. We needed to use different datasets and different study designs to address each of the posed research questions. The EAVE II collaboration has already published a number of separate research papers on uptake,,,This study has several implications for policy, practice and research. So far, the achieved rates of uptake are high, but the rollout of vaccines to CYP started considerably later than in the adults. The study also showed that the BNT162b2 vaccine in children is both safe and effective in preventing symptomatic infection, but also that this protection rapidly wanes. This complicates the decisions for both the authorities, parents and their children. The most recent studies have confirmed our observations of limited effectiveness and relatively rapid waning of the 2-dose BNT162b2 COVID-19 vaccine in CYP 12–17 years of age.,,There is a clear incentive to enable children to safely return to school and that children's regular school attendance is ensured.A.S., C.R. and I.R. conceived the idea for this study. Analysis was undertaken by T.M. and K.A. IR wrote the draft of the paper and compiled supplementary online materials. Z.G., L.F., C.S., A.B., R.W., L.W., O.V.S., J.L.K.M., L.A.C., E.M., F.H., F.A., J.McM., U.A., S.A.S., S.K., C.R.S., S.V.K., and L.D.R. critically revised the manuscript, added comments and approved the final version for submission. C.R., T.M. and K.A. accessed and verified all the data. AS was responsible for the decision to submit the manuscript.https://github.com/EAVE-II/EAVE-II-data-dictionary. All code used in this study is publicly available at https://github.com/EAVE-II/Covid-Vaccines-CYP. The data used to undertake this analysis are not publicly available because they are based on deidentified national clinical records. These data are available, subject to approval by the NHS Scotland Public Benefit and Privacy Panel, by application through the Scotland National Safe Haven. The data used in this study are sensitive and will not be made publicly available.A data dictionary covering the datasets used in this study can be found at A.S., C.R. and J.McM. are members of the Scottish Government Chief Medical Officer's COVID-19 Advisory Group and A.S. of its Standing Committee on Pandemics. A.S. & J.McM. are also members of the New and Emerging Respiratory Virus Threats Advisory Group (NERVTAG) Risk Stratification Subgroup. J.McM. is the Chair of the multidisciplinary Scottish COVID-19 National Incident Management Team. A.S. is a member of AstraZeneca's Thrombotic Thrombocytopenic Taskforce. SVK is a member of the U.K. Government's Scientific Advisory Group on Emergencies subgroup on ethnicity, the Cabinet Office's International Best Practice Advisory Group and was co-chair of the Scottish Government's Expert Reference Group on Ethnicity and COVID-19. C.R. reports grants from the MRC and Public Health Scotland, during the conduct of the study, and is a member of the Scientific Pandemic Influenza Group on Modelling, Medicines and Healthcare products Regulatory Agency, Vaccine Benefit and Risk Working Group. I.R. is a member of the Scientific Council on COVID-19 of the Republic of Croatia and co-Editor-in-Chief of the Journal of Global Health. All roles have been unremunerated. All other authors report no potential competing interest.


Immediately after preparation, the L-SNEDDS and S-SNEDDS formulations were tested for amorphicity by differential scanning calorimetry. Furthermore, solubility and dissolution tests were performed. In addition, the storage stability was investigated at 30 °C/65% RH over a period of three and six months, respectively. In all cases, amorphous formulations were obtained and, especially for the model drug celecoxib, S-SNEDDS were developed that maintained the rapid and complete drug release of the underlying L-SNEDDS even over an extended storage period. Overall, the data obtained in this study suggest that the presented S-SNEDDS approach is very promising, provided that drug-loaded L-SNEDDS are co-processed with a suitable polymeric carrier. In the case of celecoxib, the E-173 variant of the novel ModE copolymer proved to be a novel polymeric carrier with great potential for application in S-SNEDDS. The presented approach will, therefore, be pursued in future studies to establish S-SNEDDS as an alternative formulation to other amorphous systems.The present study focused on a new formulation approach to improving the solubility of drugs with poor aqueous solubility. A hot melt extrusion (HME) process was applied to prepare drug-loaded solid self-nanoemulsifying drug delivery systems (S-SNEDDS) by co-extrusion of liquid SNEDDS (L-SNEDDS) and different polymeric carriers. Experiments were performed with L-SNEDDS formulations containing celecoxib, efavirenz or fenofibrate as model drugs. A major objective was to identify a polymeric carrier and process parameters that would enable the preparation of stable S-SNEDDS without impairing the release behavior and storage stability of the L-SNEDDS used and, if possible, even improving them further. In addition to commercially available (co)polymers already used in the field of HME, a particular focus was on the evaluation of different variants of a recently developed aminomethacrylate-based copolymer (ModE) that differed in M Poor water solubility is one of the most important reasons for the limited and variable oral bioavailability of drugs . While a® or Fortovase® [In recent decades, several formulation strategies have been developed that can increase the apparent solubility of poorly water-soluble drugs in gastrointestinal fluids and, provided they are drugs that can readily permeate the intestinal mucosa, also enhance oral bioavailability . The indrtovase® ,9. Howevrtovase® ,10,11. Irtovase® ,10,12. Vrtovase® ,8,13. Atrtovase® .Hot melt extrusion (HME) may be another option for S-SNEDDS production . HME is g) of the respective polymer(s) is required. Due to the thermal input when aiming to formulate S-SNEDDS via HME, the process temperature might be an essential parameter. It will need to be kept below the decomposition temperatures of the individual components in order to prevent changes in material properties, the formation of toxic by-products and the possible loss of active ingredients. In addition to this, the HME of a multicomponent system containing solid and liquid components can be challenging because premature leakage of the liquid components from the matrix during the HME process must be avoided to ensure the correct mixing ratio of the individual components and their uniform distribution in the final solid product. For the polymeric material used in the HME process, in addition to Tg [w) [A successful HME process requires the input of both thermal and mechanical energy. To enable the conversion of the solid polymeric material into a flowable melt, a process temperature above the glass transition temperature  ,20, absow [The present study aimed to develop drug-loaded S-SNEDDS by the co-extrusion of L-SNEDDS and a polymeric carrier using an HME process. Experiments were performed with celecoxib-, efavirenz-, and fenofibrate L-SNEDDS formulations established in earlier studies . A furthw .The qualitative and quantitative composition of celecoxib-, efavirenz-, and fenofibrate L-SNEDDS formulations prepared by applying the screening approach developed in a preceding study  is shownAll L-SNEDDS formulations shown in After preparation and characterization of the different L-SNEDDS formulations , a varie® HPMC 100 LV was extruded at a lower screw speed than all other (co)polymers, as it would otherwise have clogged the extruder die or led to a loss of the integrity of the polymer when further increasing the extrusion temperature. Individual extrusion temperatures were used for each L-SNEDDS (co)polymer combination, while all extrusion temperatures were in the range of reported extrusion temperatures of the corresponding polymers [Affinisolpolymers ,16,23.®-based S-SNEDDS, this could be realized, whereas for reasons of processability, a slightly lower L-SNEDDS load had to be applied when working with Soluplus®. When co-processed with the Soluplus® copolymer, an L-SNEDDS load of higher than 20% (celecoxib and fenofibrate) or 16.67% (efavirenz) did not provide a solid strand. Reasons for these limitations could be the lower Tg of Soluplus® compared to all other (co)polymers processed by HME in this study, as well as a possibly insufficient binding capacity for oily formulations. However, since the manufacture of S-SNEDDS via HME is largely unexplored, this can only be speculated since data for comparing the process parameters as well as the properties of obtained S-SNEDDS is lacking.In order to enable a good comparison of formulations, the same L-SNEDDS load and drug content was targeted for each individual S-SNEDDS formulation. Except for the Soluplusg could be measured for all S-SNEDDS (g (31 °C), which was close to the storage temperature (30 °C) chosen for the stability studies, was determined for the Soluplus®-based fenofibrate S-SNEDDS. The chosen storage conditions might, therefore, allow for increased movement of the polymer chains, which could result in higher mobility of the respective drug combined with an increased risk of (re-)crystallization and thus result in lower drug release.Immediately after processing, the thermal characterization of celecoxib- , efavireS-SNEDDS . The lowThe saturation solubility  of celecoxib, efavirenz, and fenofibrate in water was determined for the pure drugs and all L-SNEDDS and S-SNEDDS . Since tp < 0.05) improve the solubility of the model drugs used by preparing L-SNEDDS. The results also indicate that by processing L-SNEDDS to S-SNEDDS, it may be possible to further improve the solubility of the drug compounds. However, whether such an effect is achieved depends very much on the composition of the respective formulation. S-SNEDDS based on all commercial polymers used in the study resulted in improved solubility for all drugs compared to that obtained with the underlying L-SNEDDS formulation. For celecoxib and fenofibrate, the largest solubility-enhancing effect was observed for S-SNEDDS based on Affinisol® HPMC 100 LV, whereas for efavirenz S-SNEDDS, this was the case for the Soluplus®-based S-SNEDDS polymers.To get a first impression of the drug release behavior of L- and S-SNEDDS formulations, dissolution studies of drug-loaded L- and S-SNEDDS were performed in USP apparatus II using 500 mL of 0.1 M hydrochloric acid (HCl) as a dissolution medium. For comparative purposes, corresponding doses of the unprocessed drug substances were also investigated in the same test setup.® copolymer  (® (~85%), Affinisol® HPMC 100 LV (~80%), and Kollidon® 17 PF (~76%) also released a large proportion of the contained fenofibrate dose within the test duration of 120 min  b. Withineleased) a. S-SNED 120 min b. Overalw or a higher Tg of the ModE copolymer used resulted in a decrease, both in the rate and extent of celecoxib-, efavirenz-, and fenofibrate release within the test period of 120 min  was also determined by Knopp et al. [w was accompanied by decreasing viscosity and increasing flexibility of polymeric chains [w observed in the present study was in good agreement with that observed in a previous study [p < 0.05) better in vitro release performance than the ASD formulations. This is particularly clear from the example of fenofibrate, where the E-173 kDa ASD formulation released 30% [A comparison of the rate and extent of drug release of the S-SNEDDS prepared from different ModE types showed that a higher M 120 min a. These p et al. , who stuc chains . These ec chains . Concludus study . In bothased 30%  and the This first set of dissolution studies was performed using 0.1 M HCl as the dissolution medium to simulate the pH conditions of the fasting stomach, in which the ModE copolymer is also soluble. By not adding artificial surfactants, such as sodium lauryl sulfate or polysorbate 20, to the dissolution medium, which were used in previous studies to screen drug release from formulations containing celecoxib ,26, efavComparison with other dissolution data related to S-SNEDDS prepared by HME is not (yet) possible at this time because of the lack of studies in this field. Silva et al.  have rec® were found to have larger agglomerates in samples containing the active ingredients celecoxib as well as efavirenz. After a further three months, i.e., after a total of six months of storage under the same conditions (30 °C/65% RH), very slight turbidity was observed in the L-SNEDDS. As before, most of the stored S-SNEDDS formulations could be easily shaken up again. Agglomeration was observed for the celecoxib S-SNEDDS prepared using Soluplus® and Kollidon® 17 PF. Larger agglomerates were also evident in efavirenz S-SNEDDS based on the (co)polymers Soluplus®, Kollidon® VA 64, and Affinisol® HPMC 100 LV. In the case of fenofibrate S-SNEDDS, agglomeration was observed exclusively with Soluplus®-based S-SNEDDS. There was a fairly clear correlation between the agglomeration tendency and the Tgs of the corresponding S-SNEDDS formulations, which will be discussed in the following section. Overall, the agglomeration tendency was increased when the temperature of the storage conditions and the Tg were close to each other.After three months of storage under defined and constant conditions (30 °C/65% RH), the appearance of all L-SNEDDS formulations remained unchanged regardless of the drug substance incorporated. The stored samples of most S-SNEDDS formulations showed no significant agglomeration and could be easily shaken up. Only S-SNEDDS prepared using the copolymer Soluplusg of the pure (co)polymers  were observed for celecoxib S-SNEDDS based on Kollidon® VA 64, efavirenz S-SNEDDS based on Soluplus® and E-305 kDa, and fenofibrate S-SNEDDS prepared using different ModE copolymer types polymers after six months of storage revealed that the applied storage conditions did not have any significant impact on the Tpolymers . Furtheranalyzed . In someer types . InteresAfter three months of storage at 30 °C/65% RH, there was hardly any change in the release profiles for the drug-loaded L-SNEDDS. After six months of storage, a trend toward a slightly lower amount of released dose (approx. 91–92% of the applied dose) was observed for all active ingredients used when compared with results obtained from release studies directly after the preparation of the individual drug-loaded L-SNEDDS, which could indicate potential stability issues for the L-SNEDDS under these storage conditions.After storage of the drug-loaded S-SNEDDS at 30 °C/65% RH for three and six months, most of the S-SNEDDS showed slightly different dissolution performance . Whereas® 17 PF- and the E-305 kDa-based S-SNEDDS after only three months of storage  lower amount of drug released after a test duration of 120 min compared to their release performance immediately after manufacture.For the efavirenz S-SNEDDS, a major decrease in drug release after the test duration of 120 min was noticed for E-305 kDa-based S-SNEDDS after three months of storage . Similar storage b. As obs®, Kollidon® VA 64, Kollidon® 17 PF, E-281 kDa, and Affinisol® HPMC 100 LV , d-α-Tocopherol polyethylene glycol 1000 succinate (d-TPGS), isopropyl myristate (IPM-100), and polyoxyethylene (23) lauryl ether (Brij® 35) were purchased from Sigma Aldrich Chemie GmbH . Lauroyl polyoxyl-32 glycerides (Gelucire® 44/14) and diethylene glycol monoethyl ether (Transcutol® HP) were kindly donated by Gattefossé S.A.S . Medium-chain triglycerides (Miglyol® 812) were obtained from Caesar & Loretz GmbH . Polyvinyl caprolactam–polyvinyl acetate–polyethylene glycol graft copolymer , polyvinylpyrrolidone-polyvinyl acetate copolymer  and polyvinylpyrrolidone  were purchased from BASF SE . Hydroxypropyl methylcellulose  was provided by Dow Chemical Company . Dimethylaminopropyl methacrylamide-butyl methacrylate-methyl methacrylate copolymer , a modified Eudragit® E copolymer (ModE) in different Mws, i.e., E-173 kDa, E-254 kDa, E-281 kDa, and E-305 kDa, is an in-house product of Evonik Operations GmbH  and was synthesized as described in [Celecoxib (purity of 99.7%) was obtained from Aarti Drugs Ltd. , efavirenz (purity of 99.3%) was purchased from Angene International Ltd. , and fenofibrate (purity of 99.1%) was obtained from D.K. Pharma Chem PVT Ltd. . Polyoxyethylene (80) sorbitan monooleate polymers and an L-SNEDDS formulation containing one of the poorly soluble model compounds was prepared at a predetermined mixing ratio by mixing the components in a 100-mL jar sealed with a screw cap for 10 min using a turbula mixer . Subsequently, this mixture was processed into S-SNEDDS by HME using a co-rotating HAAKE MiniLab twin screw extruder with a conical screw design . The HME process was defined by the set parameters screw speed and process temperature as well as by the torque recorded during the process. The die diameter was 2 mm, and the strand leaving the extruder was allowed to cool during transport via a conveyor belt before it was finally ground to coarse granules by a small chopper with a rotating metal gear. These coarse granules were then pulverized using a ZM 200 ultra-centrifugal mill from Retsch GmbH  (mesh size: 0.25 mm). The obtained powders were used for all subsequent experiments.Following their preparation and characterization, drug-loaded L-SNEDDS were co-extruded with different (co)polymers in individual HME processes. Marketed (co)polymers already established for the purpose of solubility enhancement, such as Soluplusg was taken from the thermogram obtained from the second heating cycle, and the indicated value represents the mean of n = 3.All S-SNEDDS formulations were analyzed via DSC (DSC 3+ (DSC-HC01), Mettler Toledo, Giessen, Germany) to determine whether the incorporated drug was in the amorphous (glass transition) or crystalline  state. The DSC method used was the same as in a preceding study . BrieflyThe saturation solubility of celecoxib, efavirenz, and fenofibrate in water was studied for the pure drug substances and the corresponding L-SNEDDS and S-SNEDDS immediately after preparation. For this purpose, about 25 mg of the drug substance or an amount of S-SNEDDS equivalent to this amount was added to a volume of 25 mL of distilled water and stirred (100 rpm) at a controlled temperature of 20 °C for 48 h. The resulting suspensions were filtered via a 0.22 µm polytetrafluoroethylene (PTFE) membrane filter (25 mm diameter) from Global Biomed Scientific . The suitability of the filter material was validated for each of the active ingredients before use. All filtrates were diluted in a specific ratio with acetonitrile before quantification of the amount of dissolved celecoxib, efavirenz, and fenofibrate by high-performance liquid chromatography (HPLC).v:v) with acetonitrile before HPLC analysis.Dissolution experiments were conducted in triplicate with 25 mg drug substance or an equivalent amount of L-SNEDDS or S-SNEDDS using USP apparatus II . The paddle speed was set to 100 rpm to avoid coning effects, and all experiments were performed in 500 mL of 0.1 M hydrochloric acid at 37 ± 0.5 °C. The test duration was 120 min. All samples were withdrawn via a fraction collector, equipped with cannula filters of 10 µm pore size and, after sampling, were manually diluted in a 1:1 ratio (An Agilent 1260 Infinity HPLC system was used for the quantification of the different model drug substances. The system consisted of a quaternary pump (G1311B), an autosampler (G1329B), a column oven (G1316A), and a UV detector (G1314C), all from Agilent Technologies . The HPLC methods used to quantify celecoxib, efavirenz, and fenofibrate were the same as described by Schmied et al.  and, priv/v), adjusted to pH 3.00 with phosphoric acid. The flow rate was set to 1.8 mL/min. An injection volume of 5 µL was applied and celecoxib was detected at 254 nm. In the concentration range of 0.13–542 µg/mL, the analytical curve was linear (r2 = 0.999995). The method was found to be accurate (100.2–102.1%) and precise (CV 2.46%) with a quantification limit of 0.05 µg/mL. The total run time was 7 min [The separation of all samples containing celecoxib was achieved using a Knauer Nucleosil 100-7 C18  column maintained at 40 °C. The mobile phase consisted of an acetonitrile:water:triethylamine mixture (300:300:0.9 as 7 min .v/v). The flow rate was set to 1.5 mL/min. An injection volume of 20 µL was applied, and efavirenz was detected at 247 nm. In the concentration range of 0.13–515 µg/mL, the analytical curve was linear (r2 = 0.999894). The method was found to be accurate (101.4–103.0%) and precise (CV 4.05%) with a quantification limit of 0.05 µg/mL. The total run time was 10 min [Separation of the samples containing efavirenz was achieved on a Symmetry 300 C18  column maintained at 22 °C. The mobile phase consisted of an acetonitrile:buffer solution (disodium hydrogen phosphate/phosphoric acid adjusted to pH 3.60) mixture (290:210 v/v), adjusted to pH 2.50 with phosphoric acid. The flow rate was set to 2.0 mL/min. An injection volume of 20 µL was applied, and fenofibrate was detected at 286 nm. In the concentration range of 0.13–526 µg/mL, the analytical curve was linear (r2 = 0.999992). The method was found to be accurate (101.2–101.4%) and precise (CV 2.42%) with a quantification limit of 0.05 µg/mL. The total run time was 7 min [The separation of all samples containing fenofibrate was achieved using a Symmetry 300 C18  column maintained at 22 °C. The mobile phase consisted of an acetonitrile:water mixture , and no interference was observed at the retention time of the specific drug. Moreover, the peak area of the drug did not change in the presence of all excipients used in the study.A quantity of approximately 10 g of each L-SNEDDS and S-SNEDDS formulation was added to a 30 mL amber glass jar, closed with a screw cap, and stored at constant and controlled conditions (30 °C/65% RH) in a climatic chamber  for six months. After three and six months, S-SNEDDS were first visually inspected to check whether they could be easily fluffed up again or stuck together (formation of larger agglomerates), whereas L-SNEDDS were analyzed for separation of their constituents and precipitation of the incorporated drug substance. Subsequently, all formulations were subjected to dissolution experiments. In addition, after six months of storage, DSC analyses were performed with the S-SNEDDS formulations. All results obtained in the stability studies were compared with those obtained immediately after manufacture.p < 0.05 in brackets.All reported data originated from at least three independent experiments. Significance tests were conducted with SigmaPlot 14.0 from Systat Software GmbH  using a one-way analysis of variance (ANOVA) followed by the Holm–Sidak test. Significances are indicated with In the present study, the preparation of S-SNEDDS from L-SNEDDS and polymeric carrier materials using HME was successfully established as a novel technology concept for the preparation of solid lipid-based formulations for improving the solubility of poorly water-soluble drugs. In particular, for the model drug celecoxib, S-SNEDDS could be developed that maintained the rapid and complete drug release of the underlying L-SNEDDS even over an extended storage period. Overall, the data obtained in the study indicate that the presented S-SNEDDS approach is very promising, provided one combines L-SNEDDS with a suitable polymeric carrier. In the case of celecoxib, the E-173 variant of the new ModE copolymer was revealed to be a novel polymeric carrier with potential for application in S-SNEDDS. Overall, the results obtained in the present study indicate that the presented S-SNEDDS formulation approach represents a novel alternative for the development of solid oral dosage forms for poorly soluble drug candidates. It is believed that this formulation approach also offers great potential for the development of stable solid formulations with improved oral bioavailability for other poorly soluble drug candidates. It merits, therefore, strong pursuit, with particular emphasis on experiments that provide more clarity on the expected in vivo behavior. Overall, therefore, it is likely to see more research addressing this formulation approach in the near future.


The accuracy of available HPV molecular assays on self-samples needs to be evaluated as compared to clinician-collected samples. This pilot study aimed to investigate the BD Onclarity™ HPV assay on vaginal and first-void urine samples. Sixty-four women referred to colposcopy for cervical dysplasia performed a vaginal self-collection and provided a first-void urine sample, after informed consent. A cervical specimen was collected during the clinician examination. All samples were tested using BD Onclarity™ HPV assay on the BD Viper™ LT System. Overall positive agreement (OPA) between cervical and self-sample results was evaluated using Cohen’s kappa value (κ). Using a clinical cut-off of 38.3 Ct for HPV 16 and 34.2 Ct for other HR genotypes, compared to cervical sample, the self-collected vaginal sample OPA was 85.9%, and κ = 0.699. Without a clinical cut-off, the OPA was 95.3%, and the κ = 0.890. Data obtained comparing cervical and urine samples showed an OPA of 87.5% with a κ = 0.79 using a clinical cut-off, and an OPA of 90.6% with a κ = 0.776 without a clinical cut-off. Data showed a substantial agreement between both self-collected and clinician-collected samples. A specific clinical cut-off analysis should be considered based on type of sample analysed. Cervical cancer is one of the most important malignancies affecting women and caused about 342,000 deaths in 2020. It is well-known that this type of tumour is caused by persisting infection of high-risk human papillomavirus (hrHPV). Recently, the World Health Organization (WHO) developed a global strategy for cervical cancer elimination to be reached by 2030, and one of the points of this strategy is to reach the 70% of women screened using a high-performance test by the age of 35, and again by the age of 45 .Eighty-five percent of cervical cancer deaths occur in developing countries, where it still represents the first leading cancer death cause. Self-sampling could be an additional strategy to reach unscreened and under-screened women, especially in middle- and low-income countries.As of now, the global use of HPV self-sampling is still limited. Only 17 countries with identified screening programs recommend the use of self-sampling in primary screening or to reach non-responder women. However, the COVID-19 pandemic has accelerated worldwide self-sampling introduction, which is now considered an important strategy to increase screening coverage in the coming years .The importance of self-sampling in improving adherence to cervical cancer screening has been well documented in the last few years, especially for women not participating in prevention programs due to different socio-cultural reasons ,4. The mDifferent devices are commercially available and seem to be suitable for this purpose. However, the accuracy of clinically validated PCR-based human papillomavirus detection kits on self-samples needs to be evaluated as compared to clinician-collected samples, as already reported in a recent meta-analysis by Arbyn et al. . PreviouUrine seems to be a good, non-invasive, and more acceptable material for the detection of HPV and sexually transmitted infections ,11,12. MThe objective of this pilot study was to evaluate accuracy of the BD Onclarity™ HPV assay on self-collected vaginal and first-void urine samples as compared to clinician-collected cervical samples.The BD Onclarity™ assay has been internationally validated for liquid-phase cytology samples for use in primary HPV screening according to both the Mejer guidelines and the VALGENT genotyping protocol ,20; in tIn particular, in this pilot study, we decided to use the same BD Onclarity™ protocol that is used for cervical specimens, without making changes, in order to discover whether the same protocol could also work for different samples compared to a liquid-phase cytology sample.For this pilot study of diagnostic accuracy, a group of 64 women (mean age: 38.4 years) with a recent diagnosis of cervical dysplasia attending the Colposcopy Clinic of San Gerardo Hospital  were enrolled. The study protocol was approved by the Ethics Committee of the University of Milano-Bicocca, Monza, Italy (Protocol n. 0037320/2017 and 0086409/2018). All subjects provided written and informed consent to participate in the study. Patients were excluded in case of immunodeficiency, HIV infection, presumed or confirmed pregnancy, diagnosis of any malignancies, and/or chemotherapy in the previous 6 months.®  and a first-void urine (FVU) sample using a Colli-pee® 20 mL  before colposcopy examination. The information brochure of the study and the instructions on how to use the devices were given to all participants.All women were adequately informed about the study by Colposcopy Clinic staff and were asked to autonomously collect a vaginal self-sample using a FLOQSwab® (Copan). All physician-collected cervical and self-collected vaginal and urine samples were sent to the Clinical Microbiology Laboratory of the Department of Medicine and Surgery, University of Milano-Bicocca, Italy, for preanalytical processing within 24 h of collection.During the colposcopy examination, the gynaecologist collected one cervical sample using an L-shaped FLOQSwab®  and transported in a tube with 20 mL of PreservCyt® Solution . All samples were well shaken using vortex for 30 seconds, and 1.5 mL aliquots were made.Cervical samples were collected using an L-shaped FLOQSwab®  and transported dry to the laboratory. Each specimen was suspended in 5.5 mL of PreservCyt® Solution , and 5 aliquots were made.Vaginal self-samples were obtained using a FLOQSwab® 20 mL device allowed for capturing a first-void urine volume of 13 mL (+/− 2 mL), in a collection device containing 7 mL of preservative urine conservation medium (UCM), leading to a final volume of 20 mL (+/− 2 mL) [The Colli-Pee/− 2 mL) . Urine sThe BD Onclarity™ HPV assay , which detects 14 high-risk (HR) genotypes, provides the capability of extended genotyping through individual detection of HPV 31, 51, and 52  and pooled detection of 33/58, 35/39/68, and 56/59/66. An endogenous human beta-globin sequence is detected as a sample validity control, sample extraction, and amplification efficiency ,21,22.All samples were tested following manufacturer instructions: BD Onclarity™ uses 0.5 mL of sample  which is added to a suitable solution produced by BD (the LBC tube) to reach a final volume of 2.2 mL, of which 0.8 mL of sample is automatically taken by the instrument to perform nucleic acid extraction using the extraction chemistry developed by BD (BD FOX ™). The extracted DNA is then eluted to a final volume of 400 microliters, and 50 microliters is automatically pipetted into each of the three wells containing the dried master mix in order to perform real-time PCR.The clinical cut-off is set to be related to CIN2+ disease, and an algorithm verifies the adequacy of the sample using the amplification of the human beta-globin gene. In particular, the software interprets the amplification curves on the basis of the following threshold cycles (Ct): 38.3 Ct for HPV16, 34.2 Ct for other HR-HPVs, and 34.2 Ct for beta-globin. Samples which were judged invalid were re-tested a second time .http://graphpad.com/quickcalcs accessed on 30 October 2022). Agreement was interpreted as slight (κ < 0.200), fair (0.200 < κ < 0.401), moderate (0.400 < κ < 0.601), good (0.600 < κ < 0.801), very good (0.800 < κ < 1.000), or perfect (κ = 1.000). Sample results were evaluated using two different cut-offs: (i) clinical cut-off of 38.3 Ct (cycle threshold) for HPV 16 and 34.2 Ct for other HR genotypes, as indicated in the package insert and (ii) without the clinical cut-off (accepting positivity up to 40 Ct).The overall percentage agreement (OPA) between cervical and self-sample results was evaluated using Cohen’s kappa value (κ) using the GraphPad QuickCalcs software  was 36/43 = 83.7%; the NPA (negative percentage agreement) was 19/21 = 90.5%. The OPA was 85.9% (55/64), and the Cohen’s kappa was 0.699 (good agreement).However, differences in collection procedures, preanalytical procedures, and the nature of the investigated samples may influence HPV detection rates on self-collected samples as compared to cervical specimens. Therefore, data analysis was performed without the clinical cut-off (accepting positivity up to Ct 40). Without the clinical cut-off, the PPA between self-collected vaginal samples and cervical samples was 45/47 = 95.7%; the NPA was 16/17 = 94.1%. The OPA was 95.3% (61/64), and the Cohen’s kappa was 0.882 .Using the package insert cut-offs of 38.3 Ct for HPV 16 and 34.2 Ct for other HR genotypes for the cervical samples and a cut-off of 40 Ct for the vaginal samples, to account for the excessive dilution, the self-collected sample PPA was 43/43 = 100%; the NPA was 18/21 = 85.7%. The OPA was 95.3%, and the Cohen’s kappa was 0.890 . All results are reported in Using the clinical cut-offs, the PPA between first-void urine sample and cervical samples was 39/43 = 90.7% and the NPA 19/21 = 90.5%. The OPA was 87.5%, and the Cohen’s kappa was 0.79 (good agreement).Without any clinical cut-off, the PPA was 43/47 = 91.5%; the NPA was 13/17 = 76.5%. The OPA was 95.3%, and the Cohen’s kappa was 0.680 (good agreement).Using the package insert cut-offs of 38.3 Ct for HPV 16 and 34.2 Ct for other HR genotypes for the cervical samples and a clinical cut-off of 40 Ct for the urine samples, the PPA of self-collected urine samples was 42/43 = 97.7%; the NPA was 16/21 = 76.2%. The OPA was 90.6% (58/64), and the Cohen’s kappa was 0.776 (good agreement). The results are reported in Comparing the results obtained from hrHPV testing with the clinical data, we evaluated the overall HPV positivity considering women with abnormal colposcopy findings and women with CIN2+ biopsy. Results for the cervical, vaginal, and urine samples with and without a clinical cut-off are presented in There is presently great interest in the use of self-collected samples as an alternative strategy in cervical cancer screening. The introduction of self-sampling in screening has been demonstrated to be more acceptable to women, resulting in improved participation in prevention programs ,23.Nowadays, several devices for self-sampling are commercially available, with different performances in sample collection ,25,26. T® and first-void urine using the Colli-Pee® 20 mL device.The aim of this study was to evaluate accuracy of the BD Onclarity™ HPV assay on self-collected vaginal samples using a FLOQSwabThe results of the study showed a good overall positive agreement between self-collected specimens and clinician-collected samples, confirming data already reported in the expanded meta-analysis by Arbyn et al. ,7. RecenThe validity of urine samples has been widely debated. On the one hand, this type of sample is easy to collect, overcomes some cultural barriers, and could be useful for surveillance in young populations ; on the ® 20 mL are comparable to results for cervical and vaginal samples, confirming—as previously reported in the literature—the validity of this device for the collection and storage of first-void urine using a preservative medium [In this study, results from urine samples collected using the Colli-Peee medium ,17,18,32A limitation of the study is that the results come from samples collected from a population of women referred to colposcopy and not from a primary screening setting. However, the purpose of the study was to understand whether the BD Onclarity™ HPV assay could be used in combination with self-samples; hence, it was necessary to have a sufficient number of HPV-positive women to answer this question. For the same reason, previous studies aiming to evaluate HPV testing on clinician-collected vs. self-collected samples have been conducted in a colposcopy setting ,33,34. TIn the present study, vaginal swabs resuspended in 5.5 mL of PreservCyt and 20 mL of first-void urine samples were processed using the same preanalytical protocol as cervical samples by placing 0.5 mL of each sample’s starting volume in tubes containing 1.7 mL of BD preserve fluid. Differences in collection procedures, preanalytical procedures, and the nature of the investigated samples may influence HPV detection rates on self-collected samples as compared to cervical specimens, on which the BD Onclarity™ HPV assay protocol and clinical cut-offs had been previously validated. For this reason, we have analysed the data obtained both with the cut-offs set by the manufacturer for cervical samples and without the pre-set cut-offs, still obtaining comparable results in terms of concordance and agreement. However, the different results obtained underline the importance of considering using different testing protocols and analytical cut-offs based on the sample type.No invalid samples were observed in this pilot study after retesting, irrespective of the sample type. The good sample adequacy observed for vaginal and first-void urine samples may have resulted from collection at the point of care and to reduced time from collection to laboratory testing.HPV genotyping in self-collected samples represents another strength of the BD Onclarity™ HPV assay, as genotyping could be a good strategy for the triage of HR-HPV-positive women in order to identify those at greater risk of cervical cancer progression, such as HPV 16 and/or 18 positive women. The use of HPV genotyping assays could be very important, particularly for screening programs based on self-sampling due to the impossibility to performing cytology triage on the same sample. Moreover, HPV genotyping on self-taken samples could be helpful to look for persistence of the same HPV genotype at follow-up and as test-of-cure, without the need for a clinician-collected sample at each visit. The possibility of performing a reliable test to verify the success of surgical treatment or the risk of relapse on a self-taken sample can represent another great advantage for women to consider.Due to the low number of positive samples, we did not perform a statistical analysis of specimens’ agreement considering specific HPV genotypes. However, these preliminary results showed a good concordance, especially for HPV 16, even if a further study enrolling a larger number of patients is necessary to obtain statistically significant results.Up to now, the importance of self-sampling has been stressed to increase adherence to cervical cancer screening programs for women not participating due to socio-cultural or physical barriers. The SARS-CoV-2 pandemic has added another valuable reason to enhance the use of self-sample tests collected at home by women, thus avoiding the need to go to hospital and the risk of COVID exposure and infection .Overall, data analysis without adjustment resulted in substantial agreement between the self-collected and clinic-collected samples, with PPA values of 83.7% and 90.7% for vaginal and urine samples, respectively. With correction for excess dilution in the sample preparation, there was almost perfect agreement, with PPA values of 100% and 97.7% for vaginal and urine samples, respectively. In conclusion, data from this pilot study are promising for the employment of the BD Onclarity™ HPV assay on vaginal samples and first-void urine samples, with an accuracy almost equivalent to clinician-collected cervical samples.


National and subnational C-section rates are seldom available in low- and middle-income countries to guide policies and interventions. We aimed to describe the C-section rates at the national and subnational levels in Peru (2012-2020).Based on the Peruvian national birth registry, we quantified C-section rates at the national, regional and province levels; also, by natural regions . Using individual-level data from the mother, we stratified the C-section rates by educational level, healthcare insurance and provider. Ecologically, we studied the correlations between C-section rates and human development index (HDI), altitude above sea level, proportion of the population living in poverty and proportion of rural population.C-section rate in Peru decreased slightly from 2012 (39·7%) to 2020 (38·0%). A widening gap of C-section rates was observed through the study years among the Coast that showed higher rates and the other natural regions that showed lower rates. The rates in most of the 25 regions showed a flat trend, particularly in the last four years and some provinces showed a very low rate. The rates were highest in mothers with higher education and in users of private health insurance. Higher HDI, health facility located at lower altitude, lower poverty and urbanization were positively correlated with higher C-section rates.C-section rates in Peru are above the international recommendations. Large differences by natural region, provinces and women socioeconomic status were found. Further efforts are needed to achieve the recommended C-section rates.Academy Ter Meulen grant of the Academy Medical Sciences Fund of the Royal Netherlands Academy of Arts & Sciences (KNAWWF/1327/TMB202116), Wellcome Trust (214185/Z/18/Z), Fogarty (D43TW011502). From 1,003 hits, we identified 2 studies that evaluate the worldwide caesarean section (C-section) rates. Both studies evaluated the C-section rates in a global level, one of the studies referred to the 1990-2014 period and the other the 2010-2018 period. These studies only included country-level data and did not contain any geographical nor socioeconomic sub-national differences within each country. Worldwide analysis might hide within countries differences. This hampers efforts to identify places where interventions are needed to keep C-section rates levels suggested by international health organizations.We conducted a search in PubMed on January 5From our understanding, this is the only observational study in Peru assessing the C-section rates in Peru between 2012 and 2020, with a detailed exploration at national, regional, and provincial levels. In addition, we analysed C-section rates by natural region and by available socioeconomic variables. The rates did not change substantially between 2012 and 2020, and they remained well above the international recommended levels over time. At the subnational level, larger differences were distinguished, with the highest rates being reported in the Coast and in areas with better socioeconomic indicators.This study uniquely shows that C-section rates in Peru are well above the international recommendations. A comprehensive evaluation is needed to identify the drivers of these high rates and reasons why the rates have not decreased, including potential health system factors, health workforce and user-related factors. The fact that the rates did not substantially change in this study period, calls to revise the available policies and interventions to reduce the frequency of C-sections. The subnational differences may imply that region-specific policy actions are needed, fostered, or articulated by the national government.Alt-text: Unlabelled box,,Clinical guidelines have clear indications for caesarean section (C-section) and are aimed to prevent perinatal complications for both mothers and newborns. Performing a C-section, when it is not clearly indicated, has not shown benefits and has been associated with maternal and offspring complications and may incur in unnecessary health expenditures.Sistema de Registro del Certificado de Nacido Vivo, in Spanish). This registry covers the whole Peruvian territory and all healthcare systems .,https://www.minsa.gob.pe/portada/transparencia/solicitud/. Additionally, for all provinces in Peru we collected information on proportion of people living in poverty, proportion of people living in rural areas, human development index (HDI), and altitude above sea level from the National institute of Statistics and Computing.We used information from the Online Live Birth Certificate Registration System  run by the Ministry of Labour and provides care for 29% of the population; and the private health sector.,Peru is an upper-middle income country located in South America, hosts 32,971,846 inhabitants, and has a gross domestic product of US$ 202·0 billion in 2020.For this analysis, we included all women-child pairs from the registration system between 2012 and 2020. Of 3,394,988 live births, we conducted a complete-case analysis. To ensure data quality, the following cleaning criteria and plausibility ranges were applied: a) observations with birthweight below 500 g or above 5,500 g; b) observations with gestational age outside the range of 22-44 weeks; c) observations with delivery mode missing; and d) observations with maternal age younger than 9 years were excluded. When summarizing the results at the province level only, those provinces with fewer than 30 births were further excluded.The prevalence or rate of C-section is expressed as a percentage (%) where the numerator was the number of births by C-section and the denominator was the total number of live births. The C-section rates are described in relation to time and geographic levels, and also in relation to sociodemographic characteristics: maternal education attainment , health insurance provider , and category of the health care facility where the birth was registered, whereby category I (lowest capacity) correspond to medical centers, categories II and III (highest capacity) correspond to hospitals.First, C-section rates were summarized along with 95% confidence intervals (95%CI). We used Clopper-Pearson exact CI to estimate the confidence intervals using ProCIs package in R software. Maps and time trend plots were used to characterize the spatial and temporal patterns of the C-section rates. Second, we used equiplots to illustrate the differences in C-section rates according to maternal education, health insurance, and category of the healthcare facility. Third, scatterplots were used to explore potential relationships between C-section rates and selected determinants at the province level ; these scatterplots also showed the Kendall's tau-b correlation coefficients. All statistical analyses were performed with R version 4·0·2 . Our study has been reported according to the Strengthening the Reporting of Observational studies in Epidemiology (STROBE) guidelines.We used anonymized data that was retrieved from open access websites and the study was conducted according to the guidelines laid down in the Declaration of Helsinki. The study was approved by the Research Ethics Committee of Universidad Peruana Cayetano Heredia (UPCH), Lima, Peru (SIDISI 207860).The funders of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. All authors had full access to the data in the study. All authors collectively had final responsibility for the decision to submit for publication and vouch for the data accuracy. The authors alone are responsible for the opinions in the manuscript, which do not necessarily represent those of their institutions.Supplementary Figure 1). Overall, 48·9% of the newborns were girls; the mean gestational age was 38·7 weeks (standard deviation (SD) = 1·7); the mean maternal age was 27·8 years (SD = 6·9); 11·2% of all births occurred in private health services; and the C-section rate was 36·5% . In 2020, eight out of the ten provinces with the highest C-section rates were in the Coast, while five out of the ten provinces with the lowest (non-zero) rates were in the Highlands. A similar profile was observed throughout the study period. Moreover, some provinces showed very low C-section rates, namely Viru (Coast) that had 0·1%, Chupaca (Highlands) and Datem del Marañon (Amazon) that had 0·2% each in 2020  had the highest C-section rate (49·4% (CI 95%: 48·3%-50·5%)) yet the Recuay province (Highlands) had the lowest (non-zero) rate (2·3% (CI 95%: 0·8%-6·6%). Even though both provinces are in the same region, the rate was 21-fold higher (49·4% vs 2·3%) in Santa than in Recuay.A), decreasing slightly from 39·7% in 2012 to 38·0% in 2020. Across the study period, the C-section rates in Peru ranged between 34·6% to 39·7%. However, we identified two relevant periods: a period of around 5% decrease from 2012 to 2016 (from 39.7% to 34.6%), and a period of around 3% increase from 2016 to 2020 (from 34.6% to 38.0%). When exploring the C-section rates by natural regions, the Coast had the highest C-section rates across the study period, followed by the Highlands and then the Amazon (B). Moreover, C-section rate in the Coast increased while it decreased in the rest of the natural regions showing 14% and 17% difference with the Highlands and the Amazon, respectively, in 2020. C-section rates in most of the 25 regions in Peru showed a stagnant trend, particularly in the last four years. The top three largest increases were observed in Lambayeque (15·5% in 2014 and 33·5% in 2020), Amazonas (26·7% in 2014 and 36·3% in 2020) and Lima (39·5% in 2014 and 47·6% in 2020). Decreasing trends were less often observed. The top three largest decreases were observed in San Martin (52·9% in 2014 and 32·2% in 2020), Apurimac (34·5% in 2014 and 25·0% in 2020) and Ancash (46·0% in 2014 and 37·4% in 2020) Supplementary Figure 2-3).At the national level C-section rates were high and did not change substantially over time A, decreae Amazon B. MoreovA). Between 2012 to 2020, C-section rates amongst mothers with incomplete secondary or lower education decreased from 34·2% to 24·6%, while in mothers with complete secondary education decreased from 39·7 to 35·8%. Conversely, in mothers with any higher education C-section rates increased from 48·6% to 53·1% in the same period. C-section rates were the highest in mothers with a private health insurance and lowest in those with a public health insurance (e.g. SIS) (B). This difference increased over time (B). In private/out-of-pocket health expenditure settings, C-section rates increased from 46·2% in 2012 to 73·9% in 2020. In those with SIS insurance, C-section rates decreased from 36·4% to 29·0% in the same period. The C-section rates were highest in healthcare facility with a higher category, where C-section rates increased from 41·7% to 52·7% (C).C-section rates were higher in the group of mothers with higher education A. Betwee.g. SIS) B. This dver time B. In prito 52·7% C.Figure At the province level , provinces with higher HDI showed higher C-section rates. Conversely, provinces at high altitude, those with higher proportions of people living in poverty and those living in rural areas had lower C-section rates .Figure 4,,Our study shows that in Peru the national C-section rate in 2020 was 38%; that is, 2·5-fold times higher the international recommendations.Strengths of our study include the use of a national birth registry with information covering the period 2012-2020 and including both public and private hospitals throughout Peru. This rich database allowed the description of national and subnational trends in C-section rates. Nonetheless, limitations need to be addressed. Misclassification bias due to differences in registration procedures across provinces cannot be excluded. This is a common limitation when analysing national registries and should not substantially modify the time and geographic trends. Notably, data entry in the national registration system followed standard procedures and was performed by health professionals using national guidelines.22We did not present results for all provinces throughout the observation period. Early in the implementation of the national birth registry not all provinces had access to the registry though in the last five years we included most of the country. Even in recent years when the national birth registry included almost all provinces in Peru, it is still possible that some births were missed. For example, births occurring at home and in remote areas may have been missed. We argue that missed births from the national registry were very few and should not have introduced bias to our results. Of note, the national birth registry complements the online registration with manual entries for births occurring where internet connection is not available. Kendall's tau-b correlations are not the ideal metric to quantify the association between two variables. We used Kendall's tau-b correlations as a hypothesis-generating approach and to help identifying possible linear relationships in the scatterplots. Thus, they should not be interpreted as the magnitude of the association, let alone as implicating casual associations.The C-section rates in Peru were consistently well above the international recommendations and showed a marginal decrease during the study period. In the same lapse, the number of hospitals and gynaecology services also increased.,We observed reductions of C-section rates in few regions: San Martin, Apurimac and Ancash. We cautiously speculate that national guidelines and other regulations aimed to promote vaginal delivery , might have had some effect in these regions, but little effect in other regions where C-section rates did not change or even increased.,,Our findings are of particular relevance to low- and middle-income countries (LMICs), where two extreme situations can coexist in terms of C-sections, that is too little, too late (TLTL) and too much, too soon (TMTS).Although national guidelines with clear indications for when a C-section delivery is required have been introduced in Peru since 2004,Peru has not experienced substantial changes in the C-section rates at the national and subnational levels between 2012 and 2020. During this period, C-section rates remained high and above internationally recommended levels. A widening gap of C-section rates was observed through the study years among the Coast that showed higher rates and the other natural regions that showed lower rates. Women of higher socioeconomic status, better educated and those with higher access to public health insurance showed higher C-section rates over time. Further comprehensive and locally adapted interventions are urgently needed to reduce the high C-section rates in Peru, while further efforts are warranted in areas with low rates that are below the recommended standards.Conceptualization: all authors; data curation: HGQP, KNC-T and WCG-V; formal analysis: HGQ-P, KNC-T and WCG-V; writing original draft: HGQP and KNC-T; writing review & editing: HGQ-P, KNC-T, WCG-V, RMC-L, CT-M and LH. All authors approved the submitted version.https://www.minsa.gob.pe/portada/transparencia/solicitud/frmFormulario.asp, and from the National institute of Statistics and Computing http://iinei.inei.gob.pe/microdatos/. We provide the data herein analysed and the analysis code of data preparation, scatterplots and maps as supplementary materials to this paper.All data herein analysed are open access and can be requested from the Ministry of Health in Peru: Lancet Group takes a neutral position with respect to territorial claims in published maps and institutional affiliations.The The authors declare no competing interests.


The purpose of this study was to investigate the effects of valdecoxib on the retina in retinal ischemia-reperfusion injury (IRI) and R28 cells following oxygen-glucose deprivation/recovery (OGD/R) injury, as well as the underlying mechanisms. Immunofluorescence and Cell Counting Kit-8 (CCK-8) analyses were used to identify the proper timepoint and concentration of valdecoxib’s protective effect on the R28 cells in the OGD/R model. Hematoxylin-eosin (HE) staining and immunofluorescence were used to explore valdecoxib’s effect on the retina and retina ganglion cell (RGC) in IRI. Cell apoptosis was determined by a TUNEL Apoptosis Detection Kit and Annexin V-FITC/PI flow cytometry. The expression levels of p-PERK, transcription factor 4 (ATF4), GRP78, CHOP, cleaved caspase 3, bax and bcl-2 were measured by Western blot analyses. The valdecoxib protected the R28 cells from OGD/R injury by decreasing the cell apoptosis rate, and it exerted a protective effect on retinas in I/R injury by inhibiting RGC apoptosis. The valdecoxib pretreatment reversed the expression of p-PERK, ATF4, CHOP, GRP78, cleaved caspase 3 and bax induced by the glaucomatous model. Meanwhile, the CCT020312 reversed the valdecoxib’s anti-apoptosis effect by activating PERK-ATF4-CHOP pathway-mediated endoplasmic reticulum (ER) stress. These findings suggest that valdecoxib protects against glaucomatous injury by inhibiting ER stress-induced apoptosis via the inhibition of the PERK-ATF4-CHOP pathway. Glaucoma is the leading cause of irreversible blindness worldwide, characterized by the progressive loss of the visual field and retinal ganglion cells, as well as optic nerve damage . An estiThe pathogenesis of glaucoma is complicated and still under investigation, but evidence from both in vivo and in vitro models has suggested that ischemia-reperfusion injury, oxidative stress, inflammation, glutamate excitotoxicity, impaired microcirculation and dysfunctional immune responses may be involved in its onset ,5,6,7,8.Valdecoxib is a selective COX-2 inhibitor, which has been widely used in clinical practice for the treatment of osteoarthritis (OA) of the knees and hips ,17,18, rIn this study, we investigated valdecoxib’s effects on glaucomatous damage using an I/R rat model and an OGD/R cell model to elucidate the underlying molecular mechanisms.As a first step to explore OGD/R-mediated cell death, we detected the cell death rate at multiple time points with the OGD/R model using PI staining. PI-positive cells were identified as dead cells. The proportions of PI-positive cells at multiple time points were calculated. The highest PI-positive cell rate was observed at 2 h after OGD/R A,B. NextEight-week-old SD rats were sacrificed and their eyeballs were removed at 1, 3 or 7 days post-IRI. HE staining was performed to detect the morphological changes in the retina. We discovered that, compared to the control group, the retinas from 3 and 7 days post-IRI were markedly thinner. Additionally, lost RCGs or their disordered arrangement was observed at 3 and 7 days after IRI when compared with the control retina A,B. We nPrevious studies showed that OGD/R induced ER stress and increased activating transcription factor-4 (ATF4) and CHOP protein levels. We further examined whether the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-ATF4-CHOP pathway was inhibited in the OGD/R model after valdecoxib pretreatment. Western blotting A and itsAfter the valdecoxib’s effect on the PERK-ATF4-CHOP pathway and the cell apoptosis in the OGD/R model were demonstrated, we examined whether valdecoxib exerts a protective effect on the retina in IRI through similar mechanisms. Retina lysates were subjected to Western blot analysis, which demonstrated that the expression levels of p-PERK, ATF4 and CHOP increased in the I/R and I/R+DMSO groups compared to the control retina, while these proteins were reduced in the retinas pretreated with valdecoxib during the I/R. The expression of the ER stress protein GRP78 was consistent with the PERK-ATF4-CHOP pathway proteins A–H. The Recent studies have identified that CCT020312, as a selective activator of PERK, can activate the PERK-ATF4-CHOP signaling pathway . We examA wide variety of mechanisms are involved in the development and progression of glaucoma. ER stress has been recognized as playing a significant role in the pathology of glaucoma . ValdecoWe explored valdecoxib’s protective effect against glaucomatous models in vivo and in vitro. We found that pretreatment with valdecoxib reversed the morphological changes of RGC loss and thinner retinas caused by IRI. In addition, the expression of pro-apoptosis proteins increased in the I/R group and decreased after valdecoxib treatment. These results suggest that valdecoxib can alleviate I/R-induced glaucoma-like damage through the inhibition of apoptosis in RGCs. Similar results were observed in a cell model of glaucoma, demonstrating the key role of apoptosis in glaucoma pathology. This is consistent with other studies showing that the apoptosis of RGCs is the final common pathway in both human and experimental models of glaucoma ,29,30,31We further explored the underlying molecular mechanism of valdecoxib’s anti-apoptosis effect in glaucomatous models. ER stress-induced apoptosis has been implicated in the pathogenesis of various diseases . InhibitThis is the first study to explore valdecoxib’s function in glaucomatous models, providing a potential treatment for glaucoma. In addition, our results provide clues as to how we can better understand the mechanism of ER stress in glaucoma. However, while the experiments were performed in vivo and in vitro to detect valdecoxib’s function in glaucoma models, there were limitations to our study. We used the acute intraocular pressure elevation model (aHIOP) to mimic retinal I/R injury. Although this is one of the classic glaucoma models and it was chosen for a number of glaucoma research studies, the results we obtained in the I/R model have not yet been tested on other glaucoma models in vivo. It is uncertain whether the same results can be obtained in other glaucoma animal models. More glaucoma models need to be considered to test our outcomes.In conclusion, our study indicates that the PERK-ATF4-CHOP pathway plays a significant pathological role in glaucomatous damage. Valdecoxib protects against glaucomatous injury by inhibiting endoplasmic reticulum stress-induced apoptosis via the inhibition of the PERK-ATF4-CHOP pathway. These findings suggest a promising role for valdecoxib therapy in protecting individuals from glaucoma.2.R28 retinal neuronal-like cells were obtained from Dr. Kun Xiong  and cultured in low-glucose DMEM with 10% fetal bovine serum  and 1% penicillin–streptomycin  at 37 °C in a humidified atmosphere containing 5% COThe rats used in the retinal I/R model were 8-week-old male Sprague–Dawley rats purchased from Slaccas, Changsha, China and bred in animal facilities of Central South University . The study protocols were approved by the Animal Research Committee of the Xiangya School of Medicine . All animal experiments were carried out in accordance with the Guide for the Care and Use of Laboratory Animals . We purchased propidium iodide ; Cell Counting Kit-8 , Annexin V-FITC/PI Kit , TUNEL BrightRed Apoptosis Detection Kit , agonist: CCT020312 , primary antibody: RBPMS , GRP78 antibody , CHOP antibody , ATF4 , PERK , bax , bcl-2  and caspase 3 .We used the protocol described in our team’s previous work to build the rat I/R model . I/R injIn vivo, an anesthetized rat with dilated pupils was put under a stereomicroscope, and 3 μL of 5 μM valdecoxib was injected into the vitreous chamber using a 5-μL Hamilton syringe  30 min before the I/R model. Four concentrations  were initially chosen based on the concentration of valdecoxib we used in the OGD/R cell model and the volume of the vitreous chamber, and were confirmed empirically; 5 μM was identified as the proper concentration of valdecoxib in the glaucomatous animal model .2. For the R28 cells used to build the OGD/R model, we replaced the low-glucose DMEM with free-glucose DMEM and kept the cells under hypoxic conditions in a closed container at 37 °C for 2 h. Next, we replaced the free-glucose DMEM with low-glucose DMEM and returned the cells to the culture incubator at 37 °C and 5% COThe morphological changes to the retina induced by I/R were visualized through HE staining. We removed the rats’ eyeballs in each group and placed them in 4% paraformaldehyde for fixation, for 48 h. The eye tissue was embedded in paraffin, and 4 μm-thick slices were prepared. We followed the protocol for HE staining that was described previously .The eye tissue was embedded in paraffin and 4 μm-thick slices were prepared. We used a TUNEL BrightRed Apoptosis Detection Kit to detect the apoptosis RGCs of retinal tissue. The TUNEL positive cells’ nuclei were stained red. The slides were stained with DAPI to detect all cells’ nuclei.The Cell Counting Kit-8 assay kit was used to detect the cell viability. A microplate reader was used to detect the absorbance values at 400 nm after the cells were incubated with CCK-8 for 2 h. The cell survival rate was then calculated. g, 20 min, 4 °C). The protein concentration was detected using a commercial BCA Protein Assay Kit . Next, 30-μg of protein was loaded and separated by 10% SDS-PAGE, then transferred to a nitrocellulose (NC) membrane. After blocking with 5% bovine serum albumin (BSA) at room temperature for 2 h, the membranes were incubated with the primary antibody at 4 °C overnight. The membranes were washed three times, each for 10 min. The membranes were then incubated with the second antibody for 1 h at room temperature. The membranes were washed and the band was visualized using the imaging system and quantified by densitometry using ImageJ software.Western blotting was performed following the routine protocol. Briefly, cells and retinal tissue were obtained after treatment under the indicated conditions. The cells and retina were harvested and mixed with lysate in tubes. Then, the tubes were placed on ice for 30 min. The supernatant of the total lysates was collected after centrifugation  for 2 h. The samples were washed three times with PBS and counterstained with 1X PureBlu DAPI  for 5 min, then mounted. The slides were then viewed using a fluorescence microscope [The immunofluorescence staining process followed the routine protocol. Briefly, first, paraffin-embedded sections were deparaffinized using xylene, followed by rehydration in serial alcohol dilutions. Next, antigen retrieval with citrate buffer, permeabilization and blocking were performed. Subsequently, the sections were incubated with the primary antibody at 4 °C overnight. Next, they were washed three times, each for 10 min. The samples were incubated with the appropriate secondary antibody . SPSS 22.0 software was used to analyze all the data, and statistical comparisons were performed using one-way ANOVA.


Corona virus disease has been associated with a wide variety of fungal and bacterial co-infections. These secondary infections could be due to the irrational use of antibiotics, immunosuppressive therapy, pre-existing co morbidities, and immune modulator effects of the virus. But here, we report a very rare occurring of rhino-orbito-cerebral invasive fungal sinusitis in a malnourished child and no other co morbidities.This is a case of a 6-year-old boy with severe thinness and no other co-morbidities, with mild COVID-19 infection, during the course of illness developed rhino-orbito-cerebral invasive fungal sinusitis. The child’s mother had COVID-19 1 week prior to child’s illness. The child then developed fever followed by headache. The child reported to hospital on seventh day of illness and RTPCR for COVID-19, turned positive. The child’s vitals were stable and maintaining saturation. Child was being treated with supplements and symptomatic treatment for fever. On his second day of stay at hospital, he started to develop gradually progressive left-sided peri-orbital swelling. Due to the association of COVID-19 with fungal infection, child was started on AMPHOTERICIN-B and given for 4 days and referred to a higher center for further management. Radiological imaging was suggestive of rhino-sinusitis with orbital cellulitis with meningeal enhancement suggestive of fungal etiology. Debridement was done, child was adequately treated with anti-fungal, and the child showed significant improvement along with radiological clearing.Invasive fungal infection can occur in association with COVID-19 among malnourished pediatric age groups with no other comorbidities. The coronavirus disease 2019 infection is caused by the novel severe acute respiratory syndrome corona virus 2 . A wide Among children, the most common cause of invasive fungal infection includes Aspergillus species, followed by Candida and Mucormycosis . Acute iVery few cases have been reported in the pediatric age group with no associated co morbidities. Here, we report a case of child rhino-orbito-cerebral invasive fungal sinusitis associated with coronavirus disease, in a malnourished child. Similar such cases were reported by Turbin et al. among two adolescents with COVID-19 [A 6-year-old child hailing from a region of high incidence of COVID 19, from a farming background, came to the hospital with complaints of fever from 1 week. One week prior to the illness mother was tested positive for COVID-19. The child had significant exposure to mother. RTPCR for the child was positive. The child was admitted along with the mother in the hospital. At admission, the child was active and had no difficulty in breathing. On examination, vitals were stable, maintaining saturation, and systemic examination was within normal limits. There was no history of any past hospitalisation for any significant illness in the child and had been healthy since birth. So the child was taken to be Mild COVID-19 illness and was being given symptomatic treatment along with supplements. No steroids were being given .On the second day of admission, the child started to develop insidious onset, gradually progressing left peri-orbital swelling, tender, appeared red, and proptosis was noted. Vision was intact, but the child could barely open his eyes Fig. . Due to 2 indicating severe thinness as per WHO growth chart. Local examination revealed lemon-sized left peri-orbital swelling. Diagnostic nasal endoscopy was done, and the soft tissue was sent for KOH mount and fungal/bacterial culture. KOH mount did not show any hyphae and culture did not yield any growth. CBC was then done and showed hemoglobin of 11.4, total count 16,500 (neutrophils 70%/lymphocytes 20%), and platelet count of 5.37L. The blood sugar level was 98 mg/dL, glycated hemoglobin was 4.5, and urine ketone bodies were absent. HIV and HbSAg were non-reactive. Inflammatory markers showed C-reactive protein of 72.6 mg/L, serum ferritin of 612.7 nm/mL, lactate dehydrogenase of 358U/L, and D-dimer of 0.04gm/mL. Renal function was within normal limits.At the time of admission to our hospital, vitals were stable, weight is 17 kg, length is 122.5 cm, and BMI is 11.4 kg/mThe child was taken up for debridement surgery. Amphotericin B was continued for 22 days. Then, the child developed impaired renal function test. Hence, amphotericin B withheld and started on oral poscoconazole. At the end of 1 month, the child was clinically better with decrease of swelling noticed Fig. . Follow-Due to known incidence of acute invasive fungal infection associated with coronavirus disease along with radiological imaging suggestive of fungal infection, he was treated as the same and showed significant improvement with early debridement surgery and early institution of anti-fungal therapy.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus previously unknown to mankind. It is classified as a beta-CoV of group 2B and is the cause of a serious life-threatening disease known as coronavirus disease of 2019 (COVID-19) . SARS-Co+T and CD8+T cells [Beside the acute respiratory distress syndromes (ARDS) caused by it, COVID-19 patients were found to have immune-suppression attributed to a decrease in CD4+T cells . A compl+T cells . Thus, c+T cells  and fung+T cells . The mai+T cells .This child had mild COVID-19 illness with severe thinness and no other comorbidities, immunosuppressive state, or any other abovementioned risk factors. The fungus could not be cultured due to early institution of therapy. Similar such clinical and radiological findings have been reported in two adolescents with negative culture reports by Turbin et al. [Acute invasive fungal infection has been recently associated with COVID-19 infection, but this has been in patients with several co-morbidities, receiving high-dose steroids, usually in adult patients. But this is one such rare presentation of a malnourished child with invasive fungal infection in association with COVID-19.Acute invasive fungal infection can also occur as an isolated disease in association with COVID-19. We the treating pediatricians should be aware of the possibility of secondary invasive fungal infections in association with COVID-19, and early diagnosis and treatment of this deadly infection might reduce the morbidity and mortality.


The proposed sensor has a 3D-printed cylindrical channel inserted with four double-band electrodes and partially filled with a liquid electrolyte. The double-band electrodes were fabricated by wire winding with a ~0.1 mm anode-cathode distance under controlled tension. It allows the electrode to become any 3D coil rather than a 2D structure by microfabrication and exhibits good repeatability ( The digital tilt sensors measure the absolute inclination of the Earth’s gravitational direction. This unique ability is indispensable in applications, including apparatus leveling, solar tracking, structural monitoring, automotive safety, navigation, etc. There are various sensing mechanisms with either a solid or liquid pendulum. The solid pendulum has a better dynamic response and is minaturizable. In contrast, the liquid pendulum suits high-resolution static tilt sensing but requires a large housing to overcome the capillary forces.The common commercialized digital tilt sensors are tri-axis microelectromechanical systems (MEMS) accelerometers and liquid electrolytic tilt sensors. The former measures acceleration in three orthogonal directions and calculates the gravity direction by trigonometric operation. The latter has a container partially filled with conductive liquid and the immersing electrodes. The electrical resistance between the electrodes changes during tilting, and a bridge signal conditioning circuit can extract the tilt angle. However, an AC excitation voltage is required to prevent electrolysis, resulting in complex and costly circuitry. Various tilt sensors with different mechanisms are under research, including optical, capacitive, electric contact (tilt switch), thermal, and magnetic. The optical inclinometers received attention due to their immunity to electromagnetic interference (EMI), which suits harsh environments and provides potentially high performance. The majority are the Fiber Bragg Grating (FBG) sensors ,10,11,12Capacitive sensing is mature and widely employed in the MEMS sensor. On the other hand, researchers have developed dielectric liquid capacitive tilt sensors. However, the slow liquid motion contradicts its fast response advantage. Further, the best-reported resolution is only 0.1° , comparaThe electric contact tilt sensors extend the simple tilt switch by introducing multiple contacts ,19,20,21The thermal tilt sensor has microheaters to generate natural convection and thermistors to detect gravity direction ,25,26,27Finally, the magnetic tilt sensor ,29,30 usPrevious studies developed electrochemical tilt sensors based on the molecular electronic transducer (MET) principle ,32. The 2) and four double-band electrodes located at the radius midpoints with a 90° separation. A voltage (0.7 V) across the anode and cathode drives the reversible electrolytic reaction:Its current is described by:1 to L4, respectively. Electrodes 1 and 2 form the x-axis, while electrodes 3 and 4 form the y-axis tilt sensor. When tilted along the x-axis, L2 becomes longer than L1, the same as their reaction current. On the other hand, the L3 and L4 are equal and the same as the zero tilt situation.In 2 is longer than L1 along the x-axis, and L4 is longer than L3 along the y-axis. Note that L1 and L2 in x-axis tilt angles are the same.The current from electrodes 1 and 2 increases and decreases by The double-band electrode comprises three parallel 0.1 mm diameter wires in contact  by winding the rectangular coils on a laser-cut steel plate. First, pass the platinum wire through the hole in the steel plate and stick tape to fix the open end. Another end of the wires is held on a spool located on a wire releaser with a tunable tension, as shown in 2 in a water-ethanol mixture (1:2 ratio) with a syringe and needle through a hole on the top.For the assembly, the sensor consists of a 3D-printed rigid housing  and 3D-printed soft plugs  for inserting the prepared double-band electrodes. The soft plugs enable an electrode replacement in case of failure, such as the detachment of the Pt wires from the Kapton tape short circuit. First, coat the housing with 5 x-axis transimpedance amplifiers are equal, as the two double-band electrodes exhibit an acceptable uniformity. Meanwhile, for the y-axis, two sets of component values compensate for the electrode asymmetry. Note that it will not affect the linearity of the sensor. The output for the y-axis is:This section first presents the chronoamperometry (CA), cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) of the four platinum double-band electrodes.Then, the assembled tilt sensor tilts from −25° to +25 ° and to −25°, with a 5° step along the two sensing axes. The voltage against the tilt angle and the sensor output during experiments are shown and discussed.x-axis and y-axis, respectively. The forward curve sweeps from −25° to +25° and the backward curve is the opposite. From the linear regression, the sensitivity of the x-axis is 0.121 V/° and 0.154 V/° for the y-axis. The CA above shows that the x-axis steady-state currents are higher than the y-axis. Therefore, their sensitivity difference is due to the higher resistor value for electrode asymmetry compensation (y-axis. The asymmetry generates a large zero tilt voltage on the y-axis (2.124 V) but small for the x-axis (−0.6682 V). ensation  of the yx-axis and y-axis tilting experiments. Around half of the stepping could result in a flat output, indicating that the electrolyte surface tension and viscosity are suited for the tilt sensor application. The non-steady waveform could be due to the contact angle hysteresis from the rough 3D-printed surface and the double-band electrode. y-axis in x-axis in The spikes in the non-sensing axis data are proportional to the cross-sensitivity. The cross-sensitivity of the The previous study shows that there are always transient spikes at the beginning of the tilting , originaThe previous study shows that the most considerable hysteresis happens when reversing the tilting direction due to contact angle hysteresis . HoweverThe sensor has a low power consumption, a simple circuit, and high sensitivity. However, there are a few limitations. First, the sensor has a long startup time of more than 20 s from x-axis and the y-axis, respectively. However, unpredictable hysteresis is significant due to the rough 3D-printed housing resisting the electrolyte movement.In conclusion, this paper presents a molecular electronic transducer (MET) tilt sensor containing a 3D cylindrical housing and four double-band electrodes fabricated by wire winding. The sensor is suitable for indoor static sensing requiring low power consumption. It allows the double-band electrodes to form any 3D coil instead of a 2D structure by microfabrication. The electrochemical data show good repeatability (